CN114621956B - A drought-resistant lncRNA and its application - Google Patents

Abstract

The invention provides a gene corresponding to drought-resistant lncRNA, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1. The invention also provides the lncRNA corresponding to the transcription of the gene and application thereof. The gene corresponding to the lncRNA and the transcribed lncRNA thereof can specifically respond to drought stress reaction without influencing the growth and development of plants. The gene is further transferred into cassava, the development of the transgenic strain is not affected, the transgenic strain is more tolerant to drought stress, the content of proline in the transgenic strain is obviously improved, and the content of malondialdehyde is obviously reduced after drought stress treatment, so that the lncRNA and the gene thereof can improve the drought tolerance of plants without affecting the plant development and yield. The lncRNA and the gene thereof can be used for carrying out genetic improvement on plants in the future, and effective resources are provided for improving drought tolerance of the plants.

Description

A drought-resistant lncRNA and its application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a drought-resistant lncRNA and its application.

Background technique

Cassava is an important food and economic crop in the world. It has the advantages of drought tolerance, barren tolerance, high yield and no land competition with staple crops. Therefore, it is widely planted in more than 100 countries and regions around the world. Since the introduction of cassava in my country, cassava has been widely planted in tropical and subtropical regions of my country. The yield per mu of cassava is high, and cassava root contains a lot of starch, which is one of the main raw materials for the production of starch and industrial alcohol, amino acids, fructose, and glucose. The increasing demand for cassava starch in my country puts forward higher requirements for the yield, quality and stress resistance of cassava. At the same time, regional droughts frequently occur in southern my country. Drought can affect cassava plant morphology, photosynthetic efficiency, biomass accumulation, and the activity and content of various enzymes in the body, resulting in lower cassava quality and yield loss. This is a major challenge for cassava cultivation and production. big problem. Traditional hybrid breeding has a long screening cycle and high cost, which can no longer meet people's urgent needs for cassava varieties. With the wide application of high-throughput technology and genetic engineering technology in plant research, new technologies are used to study the drought resistance of cassava. The molecular mechanism and the cultivation of cassava germplasm resources can greatly accelerate the selection of high-quality cassava varieties.

The stationary state of plants determines the stasis of their lifestyle. Plants mainly produce organic matter and obtain the energy needed for growth and development through photosynthesis. Studies on model plants have found that under drought stress, plants can respond to stress at the physiological level through osmotic adjustment, dehydration protection, and active oxygen scavenging. Guide, regulate gene expression, regulate protein activity and respond at the molecular level. In recent years, researchers have sequenced transcriptomes of cassava varieties with different drought resistance, analyzed their response genes and screened some genes related to stress response. With the deepening of cassava research on drought stress, people have a preliminary understanding of how cassava responds to drought stress. However, they want to learn more about how cassava responds to drought stress and obtain more drought-tolerant and high-yield high-quality cassava resources. , we also need to strengthen mutual cooperation with international cassava research teams to gain a more in-depth and thorough understanding of cassava's drought resistance mechanism.

Long noncoding RNA (long noncoding RNA, lncRNA) is a type of RNA with a length greater than 200nt and no protein coding ability. Because they do not encode proteins, lncRNAs have long been misunderstood as "noise" during translation. With the in-depth study of lncRNA, it has been found that lncRNA is an important regulator in animals and plants, which can regulate gene expression at multiple levels such as transcription, translation, growth and development, and participate in abiotic stress, which has become a current research hotspot. The present invention identifies lncRNA specifically expressed under stress conditions through screening and analysis, and successfully inserts the lncRNA into the cassava genome, providing germplasm resources for the study of the function of lncRNA in cassava. By studying how lncRNA expresses and functions in cassava, we can understand and explore its role in plant drought resistance, and obtain high-quality drought-resistant cassava plants, which can provide high-quality seedlings for cassava germplasm innovation.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art and provide a drought-resistant lncRNA and its application.

The first aspect of the present invention is to provide a gene corresponding to a drought-resistant lncRNA named lncRNA-171 gene, the nucleotide sequence of which is shown in SEQ ID NO:1.

The second aspect of the present invention is to provide a drought-resistant lncRNA named lncRNA-171, the nucleotide sequence of which is shown in SEQ ID NO:2. The lncRNA-171 is the RNA transcribed from the gene corresponding to the drought-resistant lncRNA described in the first aspect of the present application after the restriction endonucleases BamH I and Sal I remove the cleavage sites.

The third aspect of the present invention is to provide a recombinant vector, which includes the original vector and the gene corresponding to the lncRNA, named LncRNA-171 gene, and its nucleotide sequence is shown in SEQ ID NO.1.

Wherein, the original vector can be a vector commonly used in the field of gene recombination, such as virus, plasmid and the like. The present invention is not limited thereto. In a specific embodiment of the present invention, the pCambia1301 vector plasmid is used as the original vector, but it should be understood that other plasmids or viruses can also be used in the present invention.

Preferably, the original vector is a pCambia1301 vector plasmid, and the gene described in the first aspect of the present invention is ligated with the pCambia1301 vector plasmid by two restriction endonucleases BamH I and Sal I.

The fourth aspect of the present invention is to provide genes as described in the first aspect of the present invention, or the lncRNA described in the second aspect of the present invention, or the recombinant vector described in the third aspect of the present invention in improving plant drought tolerance applications in receptivity.

Wherein, the gene as described in the first aspect of the present invention, or the lncRNA as described in the second aspect of the present invention, or the recombinant vector as described in the third aspect of the present invention does not affect the plant drought tolerance while improving the plant drought tolerance. growth and development.

For example, in a specific embodiment of the present invention, cassava is used for experiments, the gene described in the first aspect of the present invention, or the lncRNA described in the second aspect of the present invention, or the recombination described in the third aspect of the present invention While the carrier improves the drought tolerance of cassava, the growth and development of cassava plants and tubers are not affected.

The fifth aspect of the present invention is to provide a method for amplifying the gene described in the first aspect of the present invention, comprising the following steps: using cassava leaves as materials to extract total RNA, and using PolyT to reverse transcribe cDNA obtained as a template , using lnc171-SBamH I (SEQ ID NO: 3): CGGGATCCAACCCTAAATCTCCTTCAAC and lnc171-A Sal I (SEQ ID NO: 4): ACGCGTCGACTTACAGAGAAACCACATAGAA as primers, amplified by PCR to obtain the lncRNA corresponding to the first aspect of the present invention Gene, the sequence of which is shown in SEQ ID NO:1.

The sixth aspect of the present invention is to provide a primer pair, which consists of: lnc171-S BamH I: CGGGATCCAACCCTAAATCCTCTCTCAAC and lnc171-A Sal I: ACGCGTCGACTTACAGAGAAACCACATAGAA.

The gene corresponding to the lncRNA of the present invention (named lncRNA-171 gene) and its transcribed lncRNA (named lncRNA-171) can specifically respond to drought stress without affecting plant growth and development. The lncRNA-171 gene was further transferred to cassava to obtain cassava lncRNA-171 highly expressed strains, and it was found that compared with non-transgenic strains, the lncRNA-171 highly expressed strains had no effect on plant development, but were more sensitive to drought stress. tolerance. At the same time, the content of proline in the high-expression lines of lncRNA-171 was significantly increased, and the content of malondialdehyde (MDA) was significantly reduced after drought treatment, indicating that lncRNA-171 and lncRNA-171 genes can improve the drought tolerance of plants, And does not affect plant development and root yield. In the future, lncRNA-171 and lncRNA-171 genes can be used to genetically improve plants and provide effective resources for improving plant drought tolerance.

Description of drawings

Figure 1 shows the identification results of cassava drought-responsive lncRNA-171 expression.

Figure 2 shows the secondary structure of lncRNA-171 predicted by RNA fold software.

Fig. 3 is the screening result of lncRNA-171 transgenic cassava, wherein OE#1-6 are 6 transgenic cassava lines.

Fig. 4 is the measurement results of physiological indexes of transgenic cassava lncRNA-171 high-expression lines after stress treatment.

Figure 5 shows the identification results of downstream genes regulated by lncRNA-171.

Detailed ways

Referring to the accompanying drawings, the present invention will be further described in conjunction with specific embodiments, so as to better understand the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

1. Screening and identification of lncRNA-171

Cassava was subjected to drought stress treatment (no watering until the leaves and tops wilted and then rewatered). Samples before and after treatment were collected for transcriptome sequencing, and an lncRNA was identified and named lncRNA-171. In the qPCR detection of lncRNA-171, it was found that the expression abundance of lncRNA-171 was higher in aboveground parts such as stems, leaves and tops, and the expression level in roots was lower (Figure 1A). The cassava seedlings were subjected to drought stress again, and the expression of lncRNA-171 was detected under different drought nodes. The expression level of lncRNA-171 under drought stress was 10-20 times that of the control group. After 7 days of drought stress treatment, the expression level of lncRNA-171 gradually increased, reaching the highest expression level at 11 days (Fig. 1B).

2. Prediction of the secondary structure of lncRNA-171

Single-stranded RNA forms helical regions and loops through self-folding and pairing. The prediction of RNA secondary structure is a research focus in the field of bioinformatics. Using the principle of minimum free energy, the secondary structure of lncRNA-171 was predicted, and the results are shown in Figure 2. From the above figure, it can be seen that the secondary structure of lncRNA-171 is a multi-stem-loop hairpin structure. Among them, the structure prediction There are 4 main branches, mainly including 12 stem loops and 2 convex ring structures. The blue area is the 5' end, and the red area is the 3' end. The blue area in the secondary structure indicates a high degree of base pair matching, low free energy value, and good stability. Studies have shown that the stem-loop structure of RNA plays a crucial role in the interaction between RNA and binding proteins. Therefore, the prediction of the secondary structure of lncRNA-171 can provide an important reference for the study of its function and mechanism of action.

3. Acquisition of the gene corresponding to lncRNA (lncRNA-171 gene)

The total RNA was extracted from cassava leaves, the cDNA obtained by reverse transcription of PolyT was used as a template, lnc171-SBamH I: CGGGATCCAACCCTAAATCTCTCTCAAC and lnc171-A Sal I: ACGCGTCGACTTACAGAGAAACCACATAGAA were used as primers, and the lncRNA-171 gene was amplified by PCR method. Its sequence is shown in SEQ ID No: 1 (with restriction endonuclease cutting sites of BamH I and Sal I). The lncRNA-171 gene was compared and analyzed in the NCBI database, and no homologous genes (gene sequence similarity ≥ 80%) were found for the lncRNA-171 gene.

The PCR amplification reaction system is as follows:

PCR amplification program: 98°C for 5 minutes; 98°C for 10 seconds, 55°C for 5 seconds, 72°C for 15 seconds, 32 cycles; 72°C for 5 minutes.

4. Screening of transgenic cassava

The amplified product obtained in "3. Obtaining the gene corresponding to the lncRNA (lncRNA-171 gene)" was double-digested with BamH I and Sal I and connected to the expression vector pCambia1301 after the same endonuclease digestion, and identified by sequencing The integrity of the target sequence and the correctness of the connection sequence. The DNA sequence comprising 35s and terminator and lncRNA-171 is shown in SEQ ID NO:5.

The constructed lncRNA gene overexpression vector was transformed into cassava embryogenic callus to obtain positive transgenic cassava lines. In the qPCR validation of six transgenic cassava lines (Fig. 3A), the expression level of lncRNA-171 was the highest in OE#3 and OE#6 transgenic lines, and the relative expression levels were about 80-fold and 120-fold higher than those in the control group . It shows that lncRNA-171 is expressed at a high level in transgenic cassava, and it is detected by Southern hybridization that OE#3 and OE#6 are inserted in double copies, so the subsequent functional and phenotypic verification of lncRNA-171 mainly selects OE#3 and OE# 6 of these two lines (Fig. 3B).

5. Phenotype verification of transgenic cassava

The transgenic cassava was planted in the field. At the initial stage of cultivation (after 2 months of cultivation), there was no difference between the transgenic cassava plants and the control group. In the later stage of cultivation (after 8 months of cultivation), the number and size of tubers were no different from those of the wild type. Significant difference (Fig. 3C). It shows that lncRNA-171 does not affect the development of cassava plants and roots.

6. Determination of stress treatment and physiological indicators

The transgenic seedlings of transgenic cassava (potted cutting seedlings, 1 month old) were subjected to drought stress treatment (no watering until the leaves and tops wilted and then rewatered). Seedlings showed excellent resistance to drought stress. After drought stress, only three leaves near the root of the transgenic cassava seedlings withered. After re-watering, the plants quickly recovered and the terminal buds began to grow. Excellent tolerance was shown (Fig. 4A).

In addition, the leaves of transgenic cassava transgenic seedlings (field cutting seedlings, 8 months old) were placed at room temperature (25°C), and the water content of detached leaves were measured at different times. It was found that at the same time, the leaves of transgenic lines contained The amount of water was significantly higher than that of the wild type (Fig. 4B).

In the stress response of plants, malondialdehyde and proline play an important role. The increase of malondialdehyde content will cause damage to plant cell membranes. The high content of malondialdehyde can reflect the The damage is more serious, and at the same time proline has a certain protective effect on plant cells. The transgenic seedlings of transgenic cassava (potted cuttings, 2 months old) were subjected to drought stress treatment (no watering until the leaves and tops wilted), and the contents of proline and malondialdehyde in the leaves were determined. It was found that after drought treatment, the content of proline in the transgenic cassava was higher than that in the control group (Fig. 4C), and at the same time, the content of malondialdehyde in the transgenic plants OE#3 and OE#6 was lower than that of the wild type ( Figure 4D). The experimental results showed that the transgenic cassava was less affected by drought stress than the control group, and showed better tolerance when subjected to drought stress.

7. Cassava lncRNA-171 specifically regulates the expression of multiple protein-coding genes

In order to analyze the downstream key genes and genetic pathways regulated by lncRNA-171, we carried out drought treatment on cassava wild-type and lncRNA-171 transgenic lines (without watering until the leaves and tops wilted), and collected the plants before and after treatment. For leaves and tops, transcriptome sequencing was performed. After data analysis, we used DEseq to analyze the differential expression of downstream genes. By comparing the transgenic lines before and after treatment with the control group, we selected genes with |log2Ratio|≥1 and q<0.05 as differentially expressed downstream genes. The analysis found that the control cassava, transgenic cassava OE#3 and transgenic cassava OE#6 had a total of 1246 up-regulated genes and 987 down-regulated genes after drought stress (Fig. 5A). Under drought stress, a total of 25 genes were always up-regulated by lnc RNA171, while 49 genes were always down-regulated by lnc RNA171 (Fig. 5B). Cluster analysis of these genes revealed that they function in pathways such as secondary metabolism and stress response (Fig. 5C).

The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention shall fall within the scope of the present invention.

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Claims (8)

1. A gene corresponding to lncRNA, characterized in that it is named lncRNA-171 gene, and its nucleotide sequence is as shown in SEQID NO:1. 2. An lncRNA, characterized in that it is named lncRNA-171, and its nucleotide sequence is as shown in SEQ ID NO:2. 3. A recombinant vector, characterized in that it comprises an original vector and the gene of claim 1. 4. recombinant carrier according to claim 3, it is characterized in that, described original carrier is pCambia1301 carrier plasmid, the gene described in claim 1 and pCambia1301 carrier plasmid pass through BamH I and Sal I two restriction endonuclease double enzymes cut connection. 5. The application of the gene as claimed in claim 1 or the lncRNA as claimed in claim 2 or the recombinant vector as claimed in claim 3 or 4 in improving cassava drought tolerance. 6. application according to claim 5, is characterized in that, gene as claimed in claim 1 or lncRNA described in claim 2 or the recombinant carrier described in claim 3 or 4 are improving cassava drought tolerance Sex while not affecting the growth and development of cassava. 7. a kind of amplification method of the described gene of claim 1 is characterized in that, comprises the following steps: take cassava leaf as material to extract total RNA, carry out the cDNA that reverse transcription obtains with PolyT as template, use lnc171-S BamH I: CGGGATCCAACCCTAAATCCTCTTCAAC and lnc171-A Sal I: ACGCGTCGACTTACAGAGAAACCACATAGAA as primers, amplified by PCR method to obtain the gene corresponding to the lncRNA described in claim 1, its sequence is shown in SEQ ID No:1. 8. A pair of primers, characterized in that it consists of: lnc171-S BamH I: CGGGATCCAACCCTAAATCTCTCTCAAC and lnc171-A Sal I: ACGCGTCGACTTACAGAGAAACCACATAGAA.

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